Oseltamivir Phosphate
Oseltamivir phosphate (sold by Hoffman la Roche under the trade name Tamiflu) is the prodrug form of the known viral neuraminidase inhibitor oseltamivir carboxylate, which is used in the treatment and prophylaxis of influenza and other similar viruses. Oseltamivir phosphate is itself not effective as an antiviral; it is the ethyl ester prodrug of the active antiviral agent oseltamivir carboxylate. The oseltamivir phosphate which is administered orally for use as an antiviral, is metabolized in the liver by the carboxyesterase enzyme to the active anti-viral form. Oseltamivir is a competitive inhibitor of sialic acid found on the surface proteins of normal host cells. The antiviral agent works by blocking the activity of the viral neuraminidase enzyme, preventing new viral particles from being released by infected cells.
Methods of preparing oseltamivir and derivatives or analogues thereof, have been described in the patent literature, for example, in PCT publications WO 2009/137916 (hereinafter '916) to Hudlicky et al. [1] and WO 2011/047466 (hereinafter '466) to Hudlicky et al. [2]], incorporated herein by reference. The '916 and '466 patent publications further describe intermediates useful for the process for preparing oseltamivir and derivatives thereof.
Cancer Treatment
Worldwide, millions of people die from cancer every year. The American cancer society reports that half of all men and one-third of all women in the United States will develop cancer during their lifetimes. Today millions of people are living with cancer or have had cancer. The US National Cancer Institute's Surveillance Epidemiology and End Results (SEER) study estimated cancer prevalence, in the United States in 2007 at 11,714,000.
Carcinomas of the lung, prostate, breast, colon, pancreas, and ovary have a high incidence of cancer death especially if the cancer is found at a late stage of progression. These and virtually all other carcinomas may further lead to metastatic disease which in many instances is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, there is still a risk of recurrence.
In particular, patients with pancreatic cancer often present with advanced disease that is lethal and difficult to treat. Despite routine use of chemotherapy and radiotherapy, survival rate of patients with advanced pancreatic cancer has not improved dramatically. Chemo- and radiotherapy provide little or no benefit. These outcomes demand an urgent need for novel therapeutic approaches. Consequently, the development of novel cancer treatment strategies is critically essential to improve the clinical management and prognosis of cancer patients and in particular patients with pancreatic cancer.
Research in the field of cancer treatment has looked at ways to modulate cellular pathways that are essential for cancer to survive and grow. Numerous receptors and molecular pathways have been implicated in oncogenesis and cancer growth and proliferation including Ras, EGFR, VEGF, gastrin and matrix metalloproteinases.
The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is the cell surface receptor member of the epidermal growth factor (EGF-family) of extracellular protein ligands. The EGFR is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and HER 4 (ErbB-4). Mutations affecting EGFR expression or activity could result in cancer.
The Ras subfamily (an abbreviation of RAt Sarcoma) is a protein subfamily of small GTPases that are involved in cellular signal transduction. Activation of Ras signaling causes cell growth, differentiation and survival.
All members of the vascular endothelial growth factor (VEGF) family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through autophosphorylation. VEGF is a signal protein produced by cells that stimulates vasculogenesis and angiogenesis.
In humans, gastrin is a hormone that stimulates secretion of gastric acid (HCl) by the parietal cells of the stomach and aids in gastric motility. It is released by G cells in the stomach, duodenum, and the pancreas. Its release is stimulated by peptides in the lumen of the stomach.
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases as well as adamalysins, serralysins, and astacins. The MMPs belong to a larger family of proteases known as the metzincin superfamily. They are capable of degrading all kinds of extracellular matrix proteins, but also can process a number of bioactive molecules. They are also known to be involved in the cleavage of cell surface receptors, the release of apoptotic ligands (such as the FAS ligand), and chemokine/cytokine inactivation [3].
Although the signalling pathways of many glycosylated receptors including EGFR, VEGF, insulin and others that have been implicated in cancer are well characterized, the parameters controlling interactions between these receptors and their ligands have remained poorly defined. A novel signalling paradigm of glycosylated receptor activation by their natural ligands has been identified [4-7].
Toll-Like Receptor (TLR)
It has been discloses that ligand-induced TOLL-like receptor (TLR) activation is controlled by Neu1 sialidase activation. Studies have shown that Neu1 is already in complex with the TOLL-like receptors, and activation is induced upon ligand binding of the natural ligands to their respective receptors. In addition, activated Neu1 specifically hydrolyzes α-2,3-sialyl residues linked to β-galactoside, which are distant from the ligand binding site. This removes steric hindrance to receptor dimerization, and leads to subsequent signalling pathways [4,6].
It has been found that the neuraminidase inhibitor, oseltamivir phosphate, specifically inhibits TLR ligand-induced Neu1 activity on the cell surface of macrophage and dendritic cells, and subsequently blocks TLR ligand induced NFkB activation, nitric oxide (NO) production and pro-inflammatory cytokines [4]. In addition, other purified neuraminidase inhibitors such as BCX-1827, DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid), zanamivir (4-guanidino-Neu5Ac2en), and oseltamivir carboxylate had a limited effect on inhibition of lypopolysaccharide (LPS) induced sialidase activity in live BMC-2 macrophage cells at 1-2 mM compared to the LPS positive control.
Other studies using recombinant soluble human sialidases have shown that oseltamivir carboxylate scarcely inhibited the activities of the four human sialidases even at 1 mM [8], while zanamivir significantly inhibited the human Neu2 and Neu3 sialidases in the micromolar range. Furthermore, Nan et al. using lysates from mature dendritic cells have found that zanamivir completely inhibited Neu1 and Neu3 sialidase activity at 2 mM [9].
Interesting it has been found that oseltamivir phosphate was the most potent compared to the other neuraminidase inhibitors in inhibiting the sialidase activity associated with TLR ligand treated live macrophage cells whereas this compound is known to be ineffective as an antiviral in vitro because its antiviral activity is achieved by its hydrolytic metabolite oseltamivir carboxylate [10].
To further elucidate the inhibitory capacity of oseltamivir phosphate and its hydrolytic metabolite oseltamivir carboxylate, the 50% inhibitory concentration (IC50) of each compound was determined by plotting the decrease in sialidase activity against the log of the agent concentration. It was shown that oseltamivir phosphate had an IC50 of 1.175 μM compared to an IC50 of 1015 μM for oseltamivir carboxylate [4]. These data clearly illustrate that oseltamivir phosphate is 1000-fold more potent than its hydrolytic metabolite in inhibiting the sialidase activity associated with TLR ligand treated live BMC-2 macrophage cells.
It is possible that oseltamivir phosphate could be transported through the cell membrane by a P-glycoprotein as described by Morimoto et al. [11], where the hydrolytic activation could be catalyzed by carboxylesterase [10]. The antiplatelet agent clopidogrel has been previously determined to inhibit the hydrolysis of oseltamivir phosphate by carboxylesterase as much as 90% [10]. To determine whether the oseltamivir phosphate is hydrolysed in the cell in this live cell assay system, live BMA macrophage cells were pre-treated with clopidogrel bisulfate at 280 μM and 500 μM for 2 min followed with 5 μg/mL of endotoxin lipopolysaccharide (LPS) in the presence or absence of 400 μM pure oseltamivir phosphate. The results indicated that the anticarboxylesterase agent clopidogrel had no effect on oseltamivir phosphate's capacity to inhibit LPS induced sialidase activity [4]. Together, these results suggest that oseltamivir phosphate is a potent inhibitor of the sialidase associated with TLR ligand treated live macrophage cells.
Tyrosine Kinase (Trk) Receptor
The role of Neu1 sialidase as an intermediate link in the initial process of ligand induced tyrosine kinase (Trk) receptor activation and subsequent cellular function has been studied [12]. It is reported that Neu1 forms a complex with glycosylated Trk receptors within the ectodomain [12], which is consistent with the previous reported results with TLR receptors [13,14].
It has been shown Neu1 is a requisite intermediate in regulating Trk activation following neurotrophin binding to the receptor. Furthermore, based on previous findings, it is predicted that Neu1, activated by neurotrophin binding to the receptor, will result in a rapid removal of α-2,3-sialyl residues linked to β-galactosides on Trk ectodomain to generate a functional Trk receptor [12]. Although there are four identified mammalian sialidases classified according to their subcellular localization [15], the sialidases classified as cytosolic (Neu2), plasma membrane bound (Neu3) [16-18] and Neu4 [19,20] are not involved in the sialidase activity associated with neurotrophin treated live Trk-expressing cells and primary cortical neurons. Additionally, the potentiation of GPCR-signaling via membrane targeting of Gai subunit proteins and matrix metalloproteinase-9 activation by ligand binding to the receptor is involved in the activation process of Neu1 sialidase on the cell surface [12].
Oseltamivir phosphate was found to be highly potent (IC50 3.876 μM) in inhibiting Neu1 activity induced by NGF treatment of live TrkA-expressing cells. The other neuraminidase inhibitors oseltamivir carboxylate and zanamivir had limited inhibitory effect on Neu1 sialidase activity associated with NGF treated live TrkA-expressing cells. It is speculated that the reason for the inhibitory potency of oseltamivir phosphate on Neu1 sialidase activity may be due to a unique orientation of Neu1 with the molecular multi-enzymatic complex that contains β-galactosidase and cathepsin A [21] and elastin-binding protein (EBP) [22], the complex of which would be associated within the ectodomain of Trk receptors. Another possibility may involve oseltamivir phosphate's direct effect on Neu1 sialidase with specificity for sialyl α-2,3 residues linked β-galactosyl linkage of TLR receptors. It has been reported that Neu1 desialylation of α-2,3-sialyl residues of TLR receptors enables receptor dimerization [14]. The data indicated that TLR ligand-induced NFκB responses were not observed in TLR deficient HEK293 cells, but were re-established in HEK293 cells stably transfected with TLR4/MD2, and were significantly inhibited by α-2,3-sialyl specific Maackia amurensis (MAL-2) lectin, α-2,3-sialyl specific galectin-1 and neuraminidase inhibitor oseltamivir phosphate but not by α-2,6-sialyl specific Sambucus nigra lectin (SNA).
Collectively, these findings suggest that Neu1 sialidase is one of the key regulators of neurotrophin-induced Trk activation to generate a functional receptor. Targeting Neu1 would be expected to be a key signalling inhibitor by blocking the NGF activation of the TrkA signal transduction pathway at the receptor level on the cell surface.
In other studies TrkA expression and kinase activity in human pancreatic cell lines PANC-1, MIA-PaCa-2 and APC-1 were shown to be directly correlated with gemcitabine chemo-resistance. It has been further shown that silencing RNA interference (siRNA) suppressed TrkA expression and kinase activity and furthermore increased gemcitabine induced, caspase-mediated apoptosis [23].
In other studies, Neu1 was found to negatively regulate lysosomal exocytosis in hematopoietic cells where it processes the sialic acids on the lysosomal membrane protein LAMP-1 [24]. On the cell surface, Seyrantepe et al. have shown that Neu1 can actually activate phagocytosis in macrophages and dendritic cells through the desialylation of surface receptors, including Fc receptors for immunoglobulin G (FcγR) [25]. Stomatos and colleagues have also shown that Neu1 on the cell surface is tightly associated with a subunit of cathepsin A and the resulting complex influences cell surface sialic acid in activated cells and the production of IFNγ [9]. Using Neu1-deficient mice, they produced markedly less IgE and IgG1 antibodies following immunization with protein antigens, which may be the result of their failure to produce IL-4 cytokine [26].
Understanding the ligand-induced EGFR activation has tremendous relevance in the fields of cancer biology and therapeutics. EGFR over-expression is often implicated in oncogenesis, where the downstream anti-apoptotic and pro-growth effects of EGFR signalling act to further reinforce a cancerous cell's strategies to survive and multiply. As such, analysis of EGFR expression and signalling is often incorporated into the clinical management of oncogenesis. For an example, EGFR over-expression is routinely used as a biomarker in the analysis of basal-like breast tumours, where it acts as a predictor of poor prognosis and a high rate of relapse and metastasis [27].
Additionally, the presence of EGFR mutants on a cell's surface can also have severe and negative effects on the cancer cell's survival. One of the major EGFR mutants implicated in an array of tumours is the EGFRvIII mutant, which contains a 267 amino acid deletion in the extracellular domain of the receptor, including 4 N-glycosylation sites [5, 28, 29]. The issues with this receptor stem from the fact that it remains constitutively active at all times, sending a continuous stream of pro-growth and division signals for the cancerous cell.
Novel cancer therapeutics have built upon this knowledge and function to inhibit the EGFR with the hope of shutting down its aberrant signalling pathways. There are two major forms of therapeutics which target the EGFR activation mechanism: the first involves the administration of high-affinity antibodies (ie. cetuximab) to competitively bind to the ligand-binding site, thus preventing ligand binding, and the second involves the administration of small-molecule inhibitors (ie. erlotinib, gefinitib) which bind to the tyrosine kinase portion of the receptor and inhibit its phosphorylation activity [30].
The PANC-1 cell line, a human carcinoma cell line derived from the pancreatic ductal epithelium, was used in an experiment to determine whether Neu1 sialidase was also playing a role in EGFR activation within a human pancreatic cancer model. The same results in this PANC-1 cell line were observed as were observed previously in the 3T3-hEGFR mouse fibroblast cell line in which EGF-stimulation of the EGFR rapidly induces Neu1 sialidase activity. Therefore, it is propose that Neu1 sialidase is essential for the ligand-induced EGFR activation mechanism and inhibition of Neu1 sialidase will inhibit EGFR [32].